GDF-5 is a member of the Bone Morphogenetic Proteins (BMP), which is a subclass of the TGF-β superfamily of proteins. GDF-5 includes several variants and mutants, including mGDF-5 first isolated from the mouse by Lee (U.S. Pat. No. 5,801,014). Other variants include MP52 (WO 95/04819), which is a human form of GDF-5, also known as hGDF-5 and LAP-4 (Triantfilou, et al. Nature Immunology 2, 338-345 (2001)); also CDMP-1, an allelic protein variant of hGDF-5 (WO 96/14335); also rhGDF-5, the recombinant human form manufactured in bacteria (EP 0955313); also rhGDF-5-Ala83, a monomeric variant of rhGDF-5; also BMP-14, a collective term for hGDF-5/CDMP-1 like proteins; also Radotermin, the international name designated by the World Health Organization; also HMW MP52's, high molecular weight protein variants of MP52; also C465A, a monomeric version wherein the cysteine residue responsible for the intermolecular cross-link is substituted with alanine; also other active single amino acid substitution mutants including N445T, L441P, R438L, and R438K. For the purposes of this application the term “GDF-5” is meant to include all variants and mutants of the protein, and rhGDF-5 is the exemplary member having 119 amino acids.
All members of the BMP family share common structural features including a carboxy terminal active domain and share a highly conserved pattern of cysteine residues that create 3 intramolecular disulfide bonds and one intermolecular disulfide bond. The active form can be either a disulfide-bonded homodimer of a single family member or a heterodimer of two different members (see Massague, et al. Annual Review of Cell Biology 6:957 (1990); Sampath, et al. Journal of Biological Chemistry 265:13198 (1990); Celeste et al. PNAS 87:9843-47 (1990); U.S. Pat. No. 5,011,691, and U.S. Pat. No. 5,266,683). The proper folding of the protein and formation of these disulfide bonds are essential to biological functioning, and misfolding leads to inactive aggregates and cleaved fragments.
The degradation and stabilization of proteins in general has been well described in the literature, and the use of excipients such as dextran, lactose, sorbitol, mannitol, sucrose and trehalose as cryoprotectants and osmoregulators are well documented (see for example reviews of protein stability by Arakawa et al, Advanced Drug Delivery Reviews, 46, 307-326 (2001), Wang, et al., International Journal of Pharmaceutics 185, 129-188 (1999), and on trehalose by Crowe, et al., Cryobiology 43, 89-105 (2001)). The use of excipients to protect lyophilized formulations of GDF-5 has also been described in USPAP 20040132653 by Ichikawa, et al., USPAP 20060286171 by Zhou, et al., and U.S. Patent Application Ser. No. 60/870,032 by Garigapati, et al. Lyophilization is a process that is commonly used and is comprised of freeze-drying a sample to remove water to yield a solid cake for storage, which can then be rehydrated at the time of use. For proteins such as GDF-5, the freezing, drying, and rehydration with water all represent separate insults and challenges to the structure and integrity of the protein.
The use of trehalose as a bulking agent in formulations for stabilizing solutions of the protein troponin has been described by Flaa, et al., in U.S. Pat. No. 6,165,981. The stabilization of antibodies using trehalose have also been described, such as by Lam, et al., in U.S. Pat. Nos. 6,171,586 and 6,991,790. These proteins share little structural similarity with GDF-5, and the use of formulations that are successful for other proteins are not necessarily predictable for use in stabilizing GDF-5.
In contrast, the preparation of a liquid solution of GDF-5 that is stable for prolonged periods of time at elevated temperatures, such as at room temperature, or even at body temperature, present a separate set of challenges that are distinctly different from those of freezing, drying, and rehydrating, as encountered in lyophilization and reconstitution. No longer are the biochemical insults to the protein structure derived from the removal of water, the crystallization of excipients, and the changes in the local microenvironment of the protein chains, their hydrogen and sulfide bonds, and their tertiary structure, but rather the challenge is from increased thermodynamic motion. This leads to an increased rate of oxidation, deamidation, hydrolysis, and cleavage of the amino acids of the protein as the predominant deactivation mechanisms, producing small fragments and an inactive parent molecule, with a lesser amount of aggregation than is commonly observed in lyophilization processes. Reversed phase high performance liquid chromatography (rp-HPLC) and size exclusion chromatography (SEC) appear to be more reliable indicators of protein purity and stability than other methods, such as electrophoresis.
Thus, the strategy and chemistry needed to stabilize a protein such as GDF-5 for a liquid solution at room temperature or above may require a different formulation than one for lyophilization. GDF-5 is not stable in solution for prolonged periods of time at 2-8° C., and is typically stored at temperatures between −60 to −80° C. GDF-5 is not soluble at neutral pH and is typically solubilized in acidic solutions, thereby increasing the potential for acid hydrolysis.
In view of the above-mentioned limitations and complications of preparing a stable GDF-5 liquid formulation for prolonged storage and use at elevated temperatures, new and effective formulations are needed.